Dermatological agent for assisting the transport of oxygen in the skin

ABSTRACT

The invention relates to a dermatological agent for assisting the transport of oxygen in the skin, a process for its preparation and the use thereof. The problem with the known dermatological agents is the inadequate supply of oxygen to the skin and the adjoining tissue. The object of the invention is therefore to get through the horny layer of the skin and the epidermis by penetration processes in order to increase the oxygen concentration in the dermal area and adjoining tissue and to activate metabolic processes. According to the invention, this is effected by means of a dermatological agent containing asymmetric lamellar aggregates, consisting of phospholipids and oxygen-laden fluorocarbon or fluorocarbon mixture, the amount of fluorocarbon being in the range from 0.2 to 100% weight/volume, in a carrier which is suitable for dermatological use. Preparation is effected by emulsification of the corresponding constituents, and use in ointments, creams, lotions, waters, alcoholic extracts, pastes, powders, gels, tinctures or on dressings and plasters or in a spray.

This is a continuation of application Ser. No. 08/360,847 filed on Dec.22, 1994 now abandoned.

The invention relates to a dermatological agent which penetrates intothe dermal area of the skin and adjoining tissue and produces animproved supply of oxygen in this region.

U.S. Pat. No. 4,366,169 (White) claims the use of fluorocarbons for thetreatment of skin injuries and wounds, in particular of burns. In thiscase the oxygen-containing fluorocarbon is put onto the skin, or ontoappropriate dressings or similar means either directly or as anemulsion. U.S. Pat. No. 4569784 (Moore) describes the preparation of agel with gas transport properties for use on the skin. The complicatedprocess consists in emulsifying a water-immiscible organic liquid, e.g.a fluorocarbon, in the presence of an emulsifier by an emulsificationprocess. A concentration process follows (e.g. ultra-centrifugation,ultrafiltration), which leads to the formation of a gel phase. In thethird step which then follows, the separation of the clear liquid fromthe pasty solid (gel phase) is effected by decantation, filtration orevaporation. This gel is applied to the skin in suitable formulations,and it acts there without, however, penetrating the horny layer.

EP-A-296661 (Borgarello) describes a fluorocarbon-containing singlephase system which can act as an isotropic or anisotropic formulation inthe cosmetic field and also as a dermatological agent as an oxygentransporter. In this case, fluorocarbons having a maximum concentrationof 50% are emulsified in water with perfluorinated emulsifiers of thealkanesulphonamide type in the presence of an aliphatic alcohol as anauxiliary emulsifier.

WO-A-08 459 describes a perfluorcarbon emulsion containing phospholipidvesicles as a blood substitute in which the phospholipid monomers arepolymerised. In WO-A-91 00110, fluorcarbon emulsions containingphospholipids are disclosed in which the phospholipids has saturatedcarbon bonds. From WO-A-92 06676, oil-filled only slightly lamellarvesicles composed of phospholipids are known, whose structurecorresponds to the customary vesicle structure. In the known processesdescribed, the compositions described, which contain fluorocarbons, acton the skin in a system-related manner at the site of their application.

The present invention is based on the object of getting through thehorny layer of the skin and the epidermis by penetration processes, inorder to increase the oxygen concentration in the dermal area and thetissue adjoining it and to activate metabolic processes.

According to the invention, the dermatological agent for assisting thetransport of oxygen in the skin consists of asymmetric lamellaraggregates which are constructed from phospholipids laving aphosphatidylcholine content of 30 to 99% by weight which in their core,in contrast to the well-known aqueous lipisomes, contain fluorocarbonsor mixtures thereof. The dermatological agent according to the inventionthus consists of asymmetric lamellar aggregates which consist ofphospholipids having a phosphatidylcholine content of 30 to 99% byweight and oxygen-laden fluorocarbon or fluorocarbon mixture, the amountof fluorocarbon being in the range from 1 to 100% w/v(w/v=weight/volume), in a carrier which is suitable for dermatologicaluse.

The lamellar aggregates are able, because of their phospholipidstructure, which is structurally and chemically very similar or ispartly identical to the cell membranes, and in combination with theiraggregate size, which can be controlled during preparation, to penetrateinto deeper-lying skin layers and to become active there. This takesplace in contrast to the invention descriptions mentioned in the priorart, which do not permit the transport of the fluorocarbons intodeeper-lying regions of the skin. The known processes are ineffective inrespect of the claimed effect.

A plurality of fluorocarbons can be employed, e.g. aliphaticstraight-chain and branched fluoroalkanes, mono- or bicyclic andoptionally fluoroalkyl-substituted fluorocycloalkanes, perfluorinatedaliphatic or bicyclic amines, bis(perfluoroalkyl)ethenes or mixturesthereof. Particularly preferred fluorocarbons are those such asperfluorodecalin, F-butyltetrahydrofuran, perfluorotributylamine,perfluorooctyl bromide, bis-fluoro(butyl)-ethene orbis-fluoro(hexyl)ethene or C₆ -C₉ -perfluoroalkanes.

The amount of fluorocarbons in this case is in the range from 1 to 100%w/v, preferably in the range from 40 to 100%. A particularly preferredrange is that from 70 to 100% w/v.

The phospholipids employed according to the invention are naturalphospholipids such as soya lecithin and egg lecithin, and also syntheticphospholipids. In these phospholipids, the content ofphosphatidylcholine is in the range from 30 to 99% by weight, inparticular 70 to 99% by weight, i.e. phospholipids laving highphosphatidyl contents are preferred.

In addition to phosphatidylcholine, lysolecithins can also be present inthe concentration range from 0.1 to 10% by weight and/or chargedphospholipids such as phosphatidylethanolamine,n-acetylphosphatidylethanolamine or phosphatidic acid in theconcentration range 0.1 to 30% by weight.

In contrast to the known aqueous liposomes (vesicles), thephospholipid-stabilised aggregates according to the invention carryhydrophobic fluorocarbons in their core, which are capable of thetransport of oxygen. Their interfacial chemical stabilisation iseffected primarily by a monolayer with inverse arrangement andsecondarily by a structure of bilayer films attached thereto. Accordingto the invention, the asymmetric lamellar aggregates therefore alwayshave at least one three-layer structure, in contrast to the knowntwo-layer vesicles. Because of the peculiarity of their structuralarrangement, these novel aggregates are designated as asymmetriclamellar oxygen carriers. Their exceptional colloid-chemical stabilitycan presumably be traced back to the lamellar structure and to thesurface charge of the aggregates. The latter can be traced back to thechoice of suitable phosphalipids or mixtures thereof of natural as wellas of synthetic origin. Primarily, phospholipids, in particularphosphatidylcholine in the said concentration range from 30 to 99%optionally in combination with lysolecithins of concentration from 0.1to 10% and/or charged phospholipids in the concentration range 0.1 to30% by weight are responsible for an advantageous action in this sense.The claimed action of the phospholipids is verified by correspondingnegative zeta potentials and by the measurement of charge densities (ontitration with a cationic polyelectrolyte).

It was possible to determine the dependence of the penetration rate andthe depth of penetration of the particle size of the aggregatesexperimentally by separate investigations in animal experiments usinglabelled encapsulated fluorocarbons. According to these experiments,relatively small particles migrate more rapidly and more deeply into theskin tissue than relatively large particles. The choice of fluorocarbonsor mixtures thereof according to their lipid solubility (represented bytheir critical solubility temperature CST in n-hexane) permits, as afurther important criterion, the regulation of the residence time in thetissue. While, e.g. perfluorotributylamine (F-TBA, CST 59° C.), with ahigh CST value and poor lipid solubility has a relatively largeresidence time, in contrast to this perfluorodecalin (PFD, CST 22° C.),but also F-butyltetrahydrofuran, F-hexane and others are releasedcorrespondingly more rapidly from the tissue. With the aid offluorocarbon mixtures, systems can be prepared specifically with thedesired CST values, i.e. lipid and membrane solubilities with respect tothe intended use.

The content of the fluorocarbons as oxygen carriers in the lamellaraggregates can vary between 1 and 100% w/v according to the intendedapplication. Suitable fluorocarbons are in particular: aliphaticstraight-chain and branched alkanes laving 6 to 12 carbon atoms, e.g.perfluorohexane, perfluorononane; mono- or bicyclic cycloalkanes whichare optionally F-alkyl-substituted, e.g. perfluoromethylcyclohexane,perfluorodecalin; aliphatic tertiary amines, n-containing polycycles,e.g. perfluorotripropylamine, perfluorotributylamine; perfluoroethers,such as aliphatic ethers and polyethers, F-alkylfurans, bicyclic andsubstituted bicyclic ethers having 2 or 3 oxygen atoms in the molecule,e.g. perfluorodihexyl ether, perfluorobutyltetrahydrofuran;perfluoroalkyl halides, e.g. perfluorooctyl bromide, perfluorohexylbromide, perfluorooctyl chloride; bis-F(alkyl)ethenes, e.g.bis-F(butyl)ethene, bis-F(hexyl)ethene.

The term "fluorocarbons" used here is understood as meaningperfluorinated or highly fluorinated carbon compounds or mixtures whichare able to transport gases such as O₂ and CO₂. Highly fluorinatedhydrocarbon compounds within the meaning of this invention are those inwhich most of the hydrogen atoms are replaced by fluorine atoms, e.g.the bis-F(alkyl)ethenes which, as far as can be detected, are chemicallyand biologically inert and thus non-toxic. This is usually achieved ifapproximately up to 90% of the hydrogen atoms are replaced by fluorineatoms. Preferred fluorocarbons within the meaning of the presentinvention are those in which at least 95% of the hydrogen atoms arereplaced, more preferably 98% and most preferably 100%.

Individual fluorine atoms can also be replaced by other halogen atomssuch as bromine or chlorine.

Suitable phospholipids are naturally occurring phospholipids such assoya or egg lecithin, and also lecithins (phospholipids) which can beprepared synthetically, which overall are known to be skin-compatible.Because of the advantageous action on the stability of the asymmetriclamellar aggregates, phospholipid mixtures having a content of 30 to 99%of phosphatidylcholine in addition to other naturally occurringaccompanying products are preferably used. The phospholipid content inthe dermatological formulation varies between 0.5 and 20, preferably 10to 20%.

The invention also relates to a process for the preparation of aphospholipid-containing dermatological agent, which consists inemulsifying phospholipids having a phosphatidylcholine content of 30 to99% by weight with a fluorocarbon or a fluorocarbon mixture which isladen with oxygen, the amount of fluorocarbon being in the range from0.2 to 100% w/v, and incorporating the asymmetric lamellar aggregateshaving a particle size from 50 to 1000 nm obtained in this way into acarrier which is suitable for dermatological use. Emulsification iseffected in the presence of water and optionally with the addition ofmonohydric or polyhydric aliphatic alcohols. Emulsification can also beeffected by preemulsification of the crude dispersion by addition of thefluorocarbon to an aqueous phospholipid solution at a temperaturecorresponding to the starting substances employed. The preemulsificationis appropriately effected at relatively high speeds of rotation, e.g.12,000 to 15,000 rpm. The actual homogenisation is then effected in ahigh-pressure homogeniser. The homogenisation can also be effected usingother known processes, for example ultrasound. The degree of energyinput into the disperse system turns out to be indirectly proportionalto the particle sizes.

Heat-sterilisation in an autoclave is possible without an effect on theparticle sizes. To avoid autoxidation processes in the unsaturated fattyacid radical of native lipids, antioxidants, e.g. α-tocopherol can beadded.

The use of phospholipids having high phosphatidylcholine contents isparticularly advantageous. These are in general between 10 and 99% byweight, preferably 30 to 99% by weight, in particular 70 to 99% byweight.

The fluorocarbons used are the abovementioned fluorocarbons orfluorocarbon mixtures in the limits indicated for these. With the aid oftheir known O₂ solubilities, the vapour pressure and the criticalsolubility temperature, the loading with oxygen and the depth ofpenetration can be adjusted by a person skilled in the art in acontrolled manner.

The mode of action of the fluorocarbon-containing asymmetric lamellaraggregates is based on the release of oxygen to undersupplied tissue viaa topical application. An efficient use is also conceivable foroxygen-undersupplied fatty tissue as well as for supplies which aredeficient due to arteriosclerosis.

The incorporation of the asymmetric lamellar aggregates as activesubstance into ointments, creams, lotions and other aqueous or alcoholicdermatological formulations is effected depending on the intended use,it being possible to vary the fluorocarbon content and thus the O₂availability within wide limits. The aggregates can be partially ladenor saturated with gaseous oxygen before incorporation into alldermatological systems, e.g. gels, pastes, powders, ointments, creamsand lotions. Even the saturation with the oxygen of the atmospheric airby means of the establishment of equilibrium which customarily takesplace according to Henry's law offers a higher oxygen capacity than allcomparable known systems.

The dermatological agent according to the invention can also be appliedto dressings, plasters, wound coverings, and other agents coming intocontact with the skin. For example, it can also be applied as a spray.

The invention will be illustrated in greater detail below by means ofexamples. In the associated drawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the critical solubility temperatures (CST) ofperfluorocarbon mixtures in n-hexane using perfluorodecalin as astarting point

FIG. 2 is a diagram of the critical solubility temperatures ofperfluorocarbon mixtures in n-hexane using F-octyl bromide as a startingpoint.

Some selected fluorocarbons and their O₂ solubility, their vapourpressure and their critical solubility temperature are shown in Table 1.Starting from these values, the desired characteristics for thepenetration of the skin with the aid of a dermatological composition canbe selected for mixtures of fluorocarbons.

                  TABLE 1                                                         ______________________________________                                                                    Vapour                                                         O.sub.2 solubility                                                                           Pressure                                                       [ml of O.sub.2 /100 ml                                                                       P.sub.37° C.                                                                    CST                                      Fluorocarbon of FC]         [mm Hg]  [°C.]                             ______________________________________                                        Perfluorooctyl                                                                             50             14       -24.5                                    bromide                                                                       Perfluorodecalin                                                                           40             12.5     22                                       bis-F(butyl)ethene                                                                         50             12.6     22.5                                     F-cyclohexylmethyl-                                                                        42             4        38.5                                     morpholine                                                                    F-tripropylamine                                                                           45             18.5     43                                       F-dihexyl ether                                                                            45             2        59                                       F-tributylamine                                                                            40             1        59                                       Perfluorodecalin-F-                                                                        40             7        42                                       tributylamine 1:1                                                             Perfluorobutyl-                                                                            52             51       29                                       tetrahydrofuran                                                               F-methylcyclohexane                                                                        57             180      8.2                                      F-hexane     58             414      20                                       ______________________________________                                    

EXAMPLE 1

50 ml of a 10% strength aqueous phospholipid solution (soya lecithin,40% phosphatidylcholine (PC)) are homogenised with ice-cooling togetherwith 80 g of a highly pure fluorocarbon mixture containing no H atoms(90% perfluorodecalin, 10% F-dibutylmethylamine, critical solubilitytemperature 26° C.) using an ultrasonic disintegrator until theparticles have a mean diameter of 244 nm. The lamellar structure of theaggregates of fluorocarbon and phospholipid can be detected from ³¹P-NMR measurements by the typical signal width as well as from electronmicrographs. The aggregate dispersion can be mixed with suitablealcohols (ethanol, propylene glycol, glycerol) for the purpose ofsterilisation without problems and without affecting its stability.Addition of 30 ml of ethanol produces sterility, the resultingdispersion having the following composition:

    ______________________________________                                                 62%  w/v fluorocarbons                                                        9.7% phospholipids                                                            19%  ethanol                                                         ______________________________________                                    

The zeta potential of minus 61 mV verifies a negative surface chargeproduced by the phospholipids with an electrostatic stabilisation of thedispersion. After saturation with gaseous oxygen, the dispersion isincorporated into a non-interacting ointment base which is compatiblewith the asymmetric lamellar aggregates.

EXAMPLE 2

18 g of lyophilised phospholipid of the composition [30% PC, 30% PE(phosphatidylethanolamine), 31% PI (phosphatidylinositol)] are dissolvedin 90 ml of sterilised water and treated with 16 ml of undenaturedethanol. Using a mechanical high-speed stirrer (Ultra-Turrax, 15,000rpm), the dispersion is stirred and at the same time perfluorodecalin(CST 22° C.) is added successively to the stirring container which istemperature-controlled at 20° C. The crude dispersion is homogenised at500 atm in an inert gas stream in a high-pressure homogeniser of theManton Gaulin type. At the start of the last but one passage,a-tocopherol acetate is added to the dispersion to 0.1% to avoidautoxidation processes and as a scavenger for free radicals in the skintissue.

The measurements carried out using the photon correlation spectrometerN-4 MD (Coultronics) confirm the presence of a unimodal particle sizedistribution and a mean particle diameter of 128 nm. Electron microscopyinvestigation using the "negative staining" method are in agreement withthis. According to ³¹ P-NMR investigations, the asymmetric lamellaraggregates are present in the unilamellar state with a zeta potential ofminus 76 mV. The composition of the dispersion is

    ______________________________________                                                 48%  w/v perfluorodecalin                                                     13%  phospholipids                                                            9%   ethanol.                                                        ______________________________________                                    

EXAMPLE 3

80 g of n-F-hexane, which is present in a mixture with itsperfluorinated isomers (CST 20° C.), were mechanically preemulsifiedwith 9.5 grams of egg yolk 3-sn-phosphatidylcholine in 47 ml ofdeionised and sterilised water under inert gas conditions with theaddition of 0.2% dl-alpha-tocopherol to give a crude dispersion. Thecrude emulsion was homogenised in a pressure homogeniser at pressures of500 atm under a suitable temperature regime and with checking of theparticle sizes. The dispersion obtained has a medium viscosity and aparticle diameter of 294 nm. After addition of 8 ml of propylene glycol,stability and sterility (microorganism count less than 100microorganisms/g) were observed at room temperature in a long-term test.Dilution, e.g. in the preparation of lotions, is possible withoutproblems without a change in important colloid-chemical parameters.

Investigations of the dispersion using a light microscope in polarisedlight indicate the presence of an isotropic single phase system, inwhich liquid-crystalline structures are non-existent.

EXAMPLE 4 In vivo detection of liposome penetration

A freshly isolated physiologically intact skin was fixed by its insideto an O₂ sensor (Clark electrode) and the epidermis wetted with an O₂-transporting dispersion containing asymmetric lamellar aggregates.Under these conditions, the electrode does not indicate an O₂ partialpressure. After a penetration period of 57 minutes, the aggregates hadreached the dermal skin section in the measuring area of the electrode.The O₂ partial pressure rose to a value of 159 mm Hg. The penetrationrate into the skin is dependent on the type and size of the aggregates.

We claim:
 1. Dermatological agent for assisting the transport of oxygeninto the skin, comprisingasymmetric lamellar aggregates, comprising (a)phospholipid having a phosphatidylcholine content of 30% to 99% byweight; and (b) oxygen-laden fluorocarbon or fluorocarbon mixture, theamount of fluorocarbon being in the range from 0.2% to 100%weight/volume; the aggregates having a skin penetration depending on thecritical solubility temperature of the selected fluorocarbon orfluorocarbon mixture, and being present in a carrier which is suitablefor dermatological use; and said asymmetric lamellar phospholipidaggregates comprising a central core of fluorocarbons surrounded by atleast three layers of phospholipid molecules wherein the layer adjacentto said central core has the lipophilic moiety of the phospholipidinteract with the fluorocarbon.
 2. Dermatological agent according toclaim 1,wherein the lamellar aggregates have an asymmetric, three-layer,structure originating from their fluorocarbon core.
 3. Dermatologicalagent according to claim 1,wherein the fluorocarbon is selected from thegroup consisting of aliphatic straight-chain fluoroalkanes, aliphaticbranched fluoroalkanes, monocyclic fluorocycloalkanes, monocyclicfluoroalkyl-substituted fluorocycloalkanes, bicyclic fluorocycloalkanes,bicyclic fluoroalkyl substituted fluorocycloalkanes, perfluorinatedaliphatic amines, perfluoroinated bicyclic amines,bis(perfluoroalkyl)ethenes, and mixtures thereof.
 4. Dermatologicalagent according to claim 3,wherein the fluorocarbon is selected from thegroup consisting of perfluorodecalin, F-butyl-tetrahydrofuran,perfluorotributylamine, perfluorooctyl bromide, bis-fluoro(butyl)etheneand C₆ -C₉ -perfluoro-alkanes.
 5. Dermatological agent according toclaim 1,wherein the amount of the fluorocarbon is in the range from 20%to 100% weight/volume.
 6. Dermatological agent according to claim1,wherein the amount of the fluorocarbon is in the range from 40% to100% weight/volume.
 7. Dermatological agent according to claim 1,whereinthe amount of the fluorocarbon is in the range from 70% to 100%weight/volume.
 8. Dermatological agent according to claim 1,wherein thephospholipid is selected from the group consisting of naturalphospholipids, synthetic phospholipids, and the mixtures thereof, theconcentration of the phospholipids being in the range from 0.5% to 20%by weight.
 9. Dermatological agent according to claim 1,wherein thephosphatidylcholine is present in an amount from 60% to 90%. 10.Dermatological agent according to claim 1,wherein the lipid fractionused, in addition to phosphatidylcholine, lysolecithins are present inthe concentration range from 0.1% to 5% by weight.
 11. Process for thepreparation of a dermatological agent for assisting the transport ofoxygen into the skin, comprising the steps ofemulsifying phospholipidshaving a phosphatidylcholine content of 30% to 99% by weight with anoxygen-laden fluorocarbon or fluorocarbon mixture, the amount offluorocarbon being in the range from 0.2% to 100% weight/volume toproduce asymmetric lamellar aggregates; and incorporating asymmetriclamellar aggregates having a particle size from 50 nm to 1000 nmobtained in this way into a dermatological carrier; and said asymmetriclamellar phospholipid aggregates comprising a central core offluorocarbons surrounded by at least three layers of phospholipidmolecules wherein the layer adjacent to said central core has thelipophilic moiety of the phospholipid interact with the fluorocarbon.12. Process according to claim 11,wherein the amount of fluorocarbon isin the range from 20% to 100% weight per volume; the amount ofphosphatidylcholine in the lipid fraction is in the range from 60% to90%.
 13. Process according to claim 12,wherein the amount offluorocarbon is in the range from 40% to 100%.
 14. Process according toclaim 11,wherein the particle size is in the range from 120 nm to 820nm.
 15. Process according to claim 11,wherein the particle size is inthe range from 140 nm to 400 nm.
 16. In a method for controlling thesupply of oxygen to the skin, the improvement which comprisesapplying tothe skin a dermatological agent system comprising an asymmetric lamellaroxygen carrier, containing phospholipids having a phosphatidylcholinecontent of 30% to 99% by weight; and a fluorocarbon, the fluorocarbonbeing in the range from 0.2% to 100% weight/volume and penetration intothe skin being controlled by means of the carrier structure of thephospholipid aggregates and the critical solubility temperature of thefluorocarbons; and the system being distributed in a dermatologicalcarrier selected from the group consisting of ointment, cream, lotion,water, paste, powder, gel, and tincture; and said asymmetric lamellarphospholipid aggregates comprising a central core of fluorocarbonssurrounded by at least three layers of phospholipid molecules whereinthe layer adjacent to said central core has the lipophilic moiety of thephospholipid interact with the fluorocarbon.
 17. The method claim16,wherein the system being applied to a dressing or a plaster.
 18. Themethod claim 16,wherein the system being applied by means of a spray.